Sphingolipid metabolites such as ceramides and sphingoid bases are important modulators of cell survival, proliferation, angiogenesis, migration, and vascular integrity. Our in vivo and in vitro studies indicate that sphingosine-1-phosphate (S1P), a naturally occurring bioactive lipid, is an essential angiogenic factor that regulates vascular endothelial cell (EC) permeability, a critical and defining feature of ALI. S1P acts extracellularly through its G-protein coupled S1P1-5 receptors and there is evidence that supports an intracellular role of S1P in calcium release and cell proliferation. S1P mediated cellular responses are regulated by its synthesis, catalyzed by sphingosine kinases (SphKs), and degradation mediated by S1P phosphatases, and S1P lyase (S1PL). In the course of our investigations, we observed that modulation of SphKs and S1PL expression in animals or human lung microvascular endothelial cells (HLMVECs) affected LPS-induced inflammatory responses and barrier function suggesting a key role for these sphingolipid metabolizing enzymes in maintenance of barrier function and integrity. In this competing renewal, we will test the hypothesis that "Modulation of intracellular S1P by SphKs and S1PL of the endothelium regulates LPS- mediated lung inflammation and barrier dysfunction". SA#1 will define the role of SphK1 and 2 and S1PL in modulating LPS-mediated inflammatory responses and barrier function in HLMVECs. We will utilize a combination of molecular, biochemical, and lipidomics approach to evaluate and quantify intracellular S1P accumulation and determine its role in LPs-induced IL-6 release and barrier dysfunction in the endothelium. SA#2 will define molecular mechanisms by which SphKs and S1PL regulate LPS-induced inflammation and barrier dysfunction in lung ECs. SA#3 will characterize ALI associated single nucleotide polymorphism (SNPs) in SphKs and S1PL and conduct SNP-specific association studies. SA#4 will determine the effectiveness of SphK activator(s) and S1PL inhibitor(s) as potential therapeutic agents in murine models of LPS-induced lung injury. Together, studies proposed in this competing renewal will greatly advance and clarify the key roles of SphKs and S1PL in ALI pathobiology and facilitate the development of novel therapeutic agents of sphingolipid metabolism in ameliorating sepsis-induced lung inflammation and injury. PUBLIC HEALTH RELEVANCE: Acute lung injury (ALI) is a devastating inflammatory lung injury which shares increased morbidity and mortality with enhanced vascular permeability as a defining feature. Sphingolipids such as sphingosine-1-phosphate (S1P), represent a novel biomarker and therapy for the vascular leak in ALI via stabilization or restoration of lung endothelial integrity. We propose to evaluate the enzymes of sphingolipid pathway(s) as modulators of S1P generation to improve management of patients with ALI.